Mitochondria produce ATP for the cell's metabolic needs through an oxygen consuming process called oxidative phosphorylation (OXPHOS). The survival of many organisms is therefore dependent on the availability of oxygen, which is directly utilized by the cytochrome c oxidase (COX) enzyme of the OXPHOS system. Organisms, tissues and cells have developed mechanisms to sense, respond and adapt to limiting oxygen levels (hypoxia). The hypoxic response involves both short-term changes in OXPHOS activities and long-term adaptive changes include changes in gene expression and metabolic reprogramming to promote glycolysis. Reduced OXPHOS potential due to limiting oxygen levels can lead to increased mitochondrial reactive oxygen species production and oxidative stress. A family of proteins termed the hypoxia-induced gene 1 (Hig1) family has been shown in diverse organisms to be up-regulated during hypoxic and oxidative stress conditions. Hig1 family members are found bacteria through eukaryotes, where they show widespread conservation through plants, fungi and animals, indicating their potential importance. Although of unknown function, the presence of the Hig1 proteins has been suggested to confer an anti-apoptotic function under hypoxic/oxidative stress conditions. We demonstrate here that the Hig1 proteins are novel components of the COX complex and their presence is required for COX enzyme activity. Moreover, we demonstrate that Hig1 proteins physically interact with subunit 3 (Cox3) prior to and following its assembly into the COX complex. Preliminary evidence that the Hig1-Cox3/COX interaction may also involve the highly conserved Cox12 (mCox6b) protein has also been obtained. We also provide evidence that a population of the Hig1 proteins is found in association with a nonCOX complex, and in a manner, which is influenced by the activity of the ADP/ATP carrier proteins. The experiments proposed here are designed to further our understanding of the Hig1 protein family and their role in modulating the OXPHOS activity and ensuring the hypoxic response pathway. We will specifically address the features of the Hig1 proteins that are required for the Cox3 interaction and further analyze the proposed relationship between the Hig1, Cox3 and Cox12 proteins. Another major objective of the proposed research is the determination of the identity of the nonCOX proteins that interact with the Hig1 protein and the analysis of the conserved region of Hig1 proposed to support these interactions. We hypothesize that the Hig1 protein act as intermediary between COX and nonCOX enzymes in the mitochondria and in doing so functions as a rheostat to modulate the activity of the COX complex in response to metabolic changes in the cell. PUBLIC HEALTH RELEVANCE: ATP, the vital energy source for the cell's diverse metabolic needs, is produced by mitochondria through the process of oxidative phosphorylation (OXPHOS), which involves oxygen consumption by the cytochrome c oxidase (COX) enzyme. The survival of many organisms is therefore dependent on oxygen availability and consequently organisms and cells have developed adaptive mechanisms to respond to and survive hypoxic conditions, which involves the OXPHOS system and the COX complex and the production of reactive oxygen species. Understanding the mitochondria's participation in the hypoxic response pathway is of fundamental importance because oxygen dysregulation and oxidative stress are hallmarks in the pathophysiology of many conditions such as cancer/tumors, ischaemic heart disease, diabetes, and neurological dysfunction.